Project Summary Oxygen and nitric oxide activate cellular signaling pathways, which are important in lung health and diseases. However, much less is known about the mechanisms by which lung cells (other than carotid bodies) sense and respond to changes in carbon dioxide (CO2) concentrations. An increase in the levels of CO2 (hypercapnia) is often a consequence of impaired gas exchange in lung diseases such as chronic obstructive pulmonary disease (COPD) and others. Approximately 17 million individuals in the US are afflicted by COPD, which now is the 3rd leading cause of overall mortality worldwide. Several studies have reported that patients with COPD and hypercapnia have worse outcomes. However, hypercapnia in patients with lung diseases is largely tolerated as there is still the notion that the effects of hypercapnia are not harmful to the lungs. Recent studies, including our own, have demonstrated that elevations in CO2 activate specific intracellular signaling pathways with adverse consequences for lung and organismal functions. We have conducted experiment using unbiased, hypotheses-generating, as well as hypotheses-driven approaches, which have generated preliminary data on the effects of hypercapnia on the lungs airways. Our preliminary results in preparation for this grant application suggest that high CO2 levels activate specific signaling pathways leading to changes in airway contractility. As such, we propose to elucidate the signaling pathways and mechanisms by which hypercapnia increases airway contractility and smooth muscle cell function via three interrelated specific aims. In experiments pertaining specific aim 1, we will determine whether hypercapnia increases airway smooth muscle contraction via activation of caspase-7 and downregulation of miR-133a-MEF2D. In studies pertaining specific aim 2, we will determine whether hypercapnia increases actin polymerization and thus airway smooth muscle contractility via RhoA and calcineurin and in studies pertaining specific aim 3, we will determine hypercapnia promotes airway remodeling by increasing ?SMA and extracellular matrix via RhoA-SRF and/or Wnt-CTGF pathway. Completion of the proposed experiments will provide novel information on signaling pathways and mechanisms by which high CO2 levels lead to lung airways hyperreactivity, which is of importance for patients with chronic lung diseases such as COPD and hypercapnia.